(The FASEB Journal. 2001;15:2099-2111.)
© 2001 FASEB
Defects of the insulin receptor substrate (IRS) system in human metabolic disorders
GIORGIO SESTI1,
MASSIMO FEDERICI*,
MARTA L. HRIBAL*,
DAVIDE LAURO*,
PAOLO SBRACCIA* and
RENATO LAURO*
Department of Experimental and Clinical Medicine, University of Catanzaro-Magna Graecia, 88100 Catanzaro, Italy; and
* Laboratory of Molecular Medicine, Department of Internal Medicine, University of Rome-Tor Vergata, 00133 Rome, Italy
1Correspondence: Dipartimento di Medicina Sperimentale e Clinica, Università di Catanzaro-Magna Graecia, Via Tommaso Campanella, 115, 88100 Catanzaro, Italy. E-mail:sesti{at}unicz.it
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ABSTRACT
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Insulin receptor substrate (IRS) molecules are key mediators in insulin
signaling and play a central role in maintaining basic cellular
functions such as growth, survival, and metabolism. They act as docking
proteins between the insulin receptor and a complex network of
intracellular signaling molecules containing Src homology 2 (SH2)
domains. Four members (IRS-1, IRS-2, IRS-3, IRS-4) of this family have
been identified that differ as to tissue distribution, subcellular
localization, developmental expression, binding to the insulin
receptor, and interaction with SH2 domain-containing proteins. Results
from targeted disruption of the IRS genes in mice have provided
important clues to the functional differences among these related
molecules, suggesting they play different and specific roles in vivo.
The available data are consistent with the notion that IRS-1 and IRS-2
are not functionally interchangeable in tissues that are responsible
for glucose production (liver), glucose uptake (skeletal muscle and
adipose tissue), and insulin production (pancreatic ß cells). In
fact, IRS-1 appears to have its major role in skeletal muscle whereas
IRS-2 appears to regulate hepatic insulin action as well as pancreatic
ß cell development and survival. By contrast, IRS-3 and IRS-4 genes
appear to play a redundant role in the IRS signaling system. Defects in
muscle IRS-1 expression and function have been reported in
insulin-resistant states such as obesity and type 2 diabetes. Several
polymorphisms in the IRS genes have been identified, but only the
Gly
Arg972 substitution of IRS-1, interacting with
environmental factors, seems to have a pathogenic role in the
development of type 2 diabetes. In contrast, polymorphisms of the other
IRS genes do not appear to contribute to type 2 diabetes.—Sesti, G.,
Federici, M., Hribal, M. L., Lauro, D., Sbraccia, P., Lauro, R.
Defects of the insulin receptor substrate (IRS) system in human
metabolic disorders.
Key Words: insulin signaling • IRS-1 • IRS-2 • IRS-3 • IRS-4 • type 2 diabetes
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INTRODUCTION
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TYPE 2 DIABETES is the most common metabolic disorder,
affecting > 5% of the population in Western countries. The
pathogenesis of type 2 diabetes is characterized by a combination of
peripheral insulin resistance and impaired insulin secretory capacity
of pancreatic ß cell. Genetic predisposition interacts with
environmental factors including diet, physical activity, and age
leading to the development of diabetes. Susceptibility to both insulin
resistance and insulin deficiency appears to be genetically determined
(1)
. In the majority of the prediabetic population, the
earliest abnormality is insulin resistance, which precedes the
development of glucose intolerance. Initially, the pancreas attempts to
compensate for insulin resistance by increasing insulin production and
secretion. When it is unable to maintain sufficient hyperinsulinemic
response, overt diabetes ensues. Despite intense investigations, genes
responsible for the development of type 2 diabetes remain in dispute.
Insulin initiates a wide variety of growth and metabolic effects by
binding to the insulin receptor and activating its intrinsic tyrosine
kinase. This event leads to phosphorylation on tyrosine residues of a
variety of docking proteins including insulin receptor substrate (IRS)
proteins (2)
. Phosphorylated IRS proteins serve as
multisite docking proteins for various effector molecules possessing
src homology 2 (SH2) domains, including phosphatidylinositol
3-kinase (PI 3-kinase) regulatory subunits (p85, p55 p50, p85, and
p55PIK), the tyrosine kinases Fyn and Csk, the
tyrosine protein phosphatase SHP-2/Syp, as well as several smaller
adapter molecules such as the growth factor receptor binding proteins
Grb-2, Crk, and Nck (3)
. Activation of these SH2 domain
proteins initiates signaling cascades, leading to the activation of
multiple downstream effectors that ultimately transmit the insulin
signal to a branching series of intracellular pathways that regulate
cell differentiation, growth, survival, and metabolism. Four members of
the IRS family have been identified that are considerably similar in
their general architecture (4
5
6
7)
. They are composed of an
NH2-terminal pleckstrin homology (PH) domain that
binds to membrane phospholipids, a phosphotyrosine binding (PTB) domain
located just COOH-terminal to the PH domain that is involved in
recognition of the asparagine-proline-glutamic
acid-phosphotyrosine (NPEpY) sequence located in the juxtamembrane
region of the insulin receptor ß subunit; and a less conserved
COOH-terminal portion with multiple potential tyrosine phosphorylation
motifs that can bind to specific SH2 domain-containing proteins.
Nevertheless, the four IRS proteins differ in some respects including
tissue distribution, developmental expression (8)
,
subcellular localization (9)
, and interaction with SH2
domain-containing proteins (10)
. These differences may
contribute to specificity in abilities of the IRS proteins to mediate
different biological signals. In light of their pivotal role in insulin
signaling, the IRS proteins have been considered candidate genes for
human metabolic disorders such as type 2 diabetes and obesity.
Here we will focus on recent advances in the understanding of the
functional roles of IRS proteins; we will also discuss results of
studies in animal models lacking IRS molecules by targeted disruption
of the IRS genes. Next, we will review data from human studies of the
pathogenetic role of IRS proteins in insulin signaling. Finally, we
will discuss the pathogenetic role of IRS amino acid polymorphisms in
the development of human metabolic disorders.
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IRS-1 STRUCTURE AND FUNCTION
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IRS-1 was the first substrate identified and represents the
prototype of the IRS family proteins (4)
. The human IRS-1
gene is localized on chromosome 2q36–37 (2)
. IRS-1
contains 21 putative tyrosine phosphorylation sites, several of which
are located in amino acid sequence motifs that bind to SH-2 domain
proteins, including the p85 regulatory subunit of PI 3-kinase, Grb-2,
Nck, Crk, Fyn, Csk, phospholipase C
, and SHP-2 (Fig. 1
) (2)
. IRS-1 contains also > 30 potential
serine/threonine phosphorylation sites in motifs recognized by various
kinases such as casein kinase II, protein kinase C, protein kinase
B/Akt, and mitogen-activated protein (MAP) kinases (2
, 4)
.
The relevance of IRS-1 in insulin/IGF-I signaling was first suggested
by in vitro studies in which IRS-1 was overexpressed or its levels were
decreased by antisense mRNA. Subsequently, it was shown that
overexpression of IRS-1 in CHO cells or 32D cells, a myeloid progenitor
cell line lacking both IRS-1 and IRS-2, enhanced mitogenetic effects of
insulin (11
12
13)
. Expression in isolated rat adipocytes of
an antisense ribozyme directed against rat IRS-1 resulted in a fourfold
decrease in the sensitivity of the dose-response curve for
insulin-stimulated translocation of glucose transporter GLUT4 to the
cell surface (14)
. Overexpression of recombinant human
IRS-1 in L6 rat myocytes induced an increase in insulin sensitivity and
responsiveness for glucose transport, translocation of GLUT1 and GLUT4,
and glycogen synthesis (15)
.
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Figure 1. Schematic diagram of human IRS-1, IRS-2, and IRS-4; the PH domain and
the PTB domain are shown. Putative binding sites for p85 regulatory
subunit of PI 3-kinase, Grb2, and SHP-2 are indicated. Solid arrows
indicate the strong binding sites dashed arrows indicate the weak
binding sites.
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The importance of IRS-1 in insulin/IGF-I signaling was further
confirmed in studies with cells or tissues isolated from knockout mice
lacking IRS-1 (16
, 17)
. Primary adipocytes from knockout
mice lacking IRS-1 showed a decrease in glucose transport and GLUT4
translocation to the plasma membrane in response to insulin
(17)
. In agreement with these results, it has been shown
in human adipocytes that IRS-1 is the main docking protein for the
binding and activation of PI 3-kinase in response to insulin
(18)
. Moreover, it has recently been reported that
differentiation of human preadipocytes to mature adipocytes was
characterized by a marked (>10-fold) increase in IRS-1 protein
expression whereas IRS-2 expression was modestly increased (>2-fold)
(19)
. These changes were associated with full development
of the human adipocyte phenotype, including increased
insulin-stimulated glucose transport capacity and increased expression
of GLUT4 (19)
. Fibroblasts derived from knockout mice
lacking IRS-1 displayed an impaired rate of proliferation in response
to IGF-I (20)
, whereas the lack of IRS-1 in brown
adipocytes derived from the same animals resulted in both impairment in
differentiation and a reduction in lipid synthesis in response to
insulin (21
, 22)
. More recent studies have shown that
IRS-1 may play also an important role in regulating insulin secretion
in pancreatic ß cells. Overexpression of IRS-1 in ßTC6-F7 or RIN
1046–38 insulinoma cell lines resulted in an increased secretory
response to glucose and glibenclamide, a second-generation sulfonylurea
(23
, 24)
. Islets from knockout mice lacking IRS-1
exhibited marked defects in insulin content and the insulin secretory
response to glucose (25)
. Taken together, these data
indicate that IRS-1 plays a key role in mediating both metabolic and
mitogenic effects of insulin in peripheral tissues such as muscle and
adipose tissue, and suggest a novel important role for IRS-1 in ß
cell function.
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IRS-2 STRUCTURE AND FUNCTION
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IRS-2 was initially identified as an alternative substrate for the
insulin receptor in animals with targeted disruption of the IRS-1 gene
(16
, 17)
. The cloned IRS-2 cDNA revealed a predicted
protein that is 100 residues longer than IRS-1 but sharing many
structural and functional characteristics with IRS-1 (Fig. 1)
(5)
. The human IRS-2 gene is localized on chromosome 13q34
(26)
. Human IRS-2 contains 22 potential tyrosine
phosphorylation sites, but only 13 are conserved in IRS-1. The amino
acid sequence identity between IRS-1 and IRS-2 is 43%, with some
domains such as the PH and PTB domains exhibiting higher degrees of
identity (65 and 75%, respectively). The COOH-terminal domains of
IRS-1 and IRS-2 are poorly conserved, displaying only 35% identity,
which arises largely from similar tyrosine phosphorylation motifs
surrounded by variable stretches of amino acid sequence. The middle of
IRS-2 possesses a unique region comprising amino acids 591–786 that
interacts specifically with the kinase regulatory loop binding (KRLB)
domain of the insulin receptor ß subunit (11)
. Because
this region is absent in IRS-1, this domain may contribute to the
signaling specificity of IRS-2. In addition, IRS-1 and IRS-2 may
regulate unique signaling pathways because of different tissue
distribution, subcellular localization, kinetics of
activation/deactivation, or specificity of interaction with downstream
effectors (8
, 10
, 27)
. For example, it has been shown that
IRS-1 and IRS-2 differ in their subcellular localization since IRS-1 is
twofold more concentrated in the intracellular membrane compartment
than in cytosol, whereas IRS-2 is twofold more concentrated in cytosol
than in the intracellular membrane compartment (28)
.
Further studies have shown that IRS-2 is dephosphorylated more rapidly
and activates PI 3-kinase more transiently than IRS-1, thus indicating
that differences in kinetics of activation may contribute to the
diversity of the insulin signaling transduced by IRS-1 and IRS-2
(28
, 29)
. Finally, IRS-1 and IRS-2 exhibit differences in
their capacity to interact with various downstream signaling elements
containing the SH2 domain (2
, 10)
. Thus, in vitro studies
have shown that IRS-1 and IRS-2 both bind PI 3-kinase, Grb-2, Crk, Fyn,
and phospholipase C, whereas only IRS-1 binds Abl and SHP-2
(2
, 10)
. In vivo studies with knockout animal models have
revealed several differences in the signaling capacity of IRS-1 and
IRS-2, as will be discussed later in detail.
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IRS-3 STRUCTURE AND FUNCTION
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IRS-3 was initially detected in rat adipocytes (30)
as a tyrosine-phosphorylated substrate of the insulin receptor. Cloning
of rat (6)
and mouse (31)
IRS-3 (rIRS-3 and
mIRS3) revealed proteins 700–800 amino acid residues shorter than
IRS-1 and IRS-2, but possessing a similar architecture comprising a PH
domain, a PTB domain, and a COOH-terminal domain. The mouse IRS-3 gene
is localized on the telomeric region of the chromosome 5G2
(31)
. The PH domain in rIRS-3 is homologous to the PH
domain in IRS-1 and IRS-2 (50% and 45% sequence identity with rat
IRS-1 and IRS-2, respectively). The PTB domain in rIRS-3 exhibits 48%
and 53% sequence identity with the corresponding domains in IRS-1 and
IRS-2, respectively. Outside of the PH and PTB domains, there is no
extended homology between IRS-3 and either IRS-1 or IRS-2. In the
carboxyl-terminal domain of IRS-3, 13 tyrosine residues are
potential sites of tyrosine phosphorylation (Table 1
) (6)
. Subsequent studies have shown that IRS-3 binds PI
3-kinase, SHP-2, Nck, and Shc, but Grb-2 and phospholipase C only
weakly (32)
. In addition to adipose tissue where IRS-3 was
originally isolated, IRS-3 mRNA is expressed in other tissues including
liver, lung, kidney, ovary, heart, fibroblasts, and ßTC-1 and ßTC-7
insulinoma cells (8
, 31)
. At a subcellular level, IRS-3 is
located mainly in the plasma membrane, where it activates PI 3-kinase
in response to insulin (9)
. However, IRS-3 does not seem
to be expressed in human cells, so its relevance in human metabolic
disorders is questionable.
In adipocytes isolated from knockout mice lacking IRS-1, 
50% of
insulin-stimulated glucose transport and GLUT4 translocation was
preserved by a signaling pathway involving IRS-3 rather than IRS-2
(33)
. Furthermore, overexpression of IRS-3 in isolated rat
adipocytes results in a supramaximal increase in the number of GLUT4
molecules translocated to the cell surface even in the absence of
insulin (34)
. In rat liver-derived HTC cells, IRS-3 showed
robust and prolonged tyrosine phosphorylation upon insulin stimulation
resulting in sustained association with the p85 regulatory subunit of
PI 3-kinase (35)
. It has also been shown that upon insulin
stimulation, the p85 subunit of PI 3-kinase associated with IRS-3 more
rapidly than with IRS-1 and IRS-2 in rat adipocytes (36)
.
The functional significance of differences in the time course between
IRS-3 and IRS-1/2 is not clear. Notably, overexpression of IRS-3 in 3T3
embryonic fibroblasts derived from knockout mice lacking IRS-1 resulted
in increased DNA synthesis stimulated by IGF-I despite the drastic
impairment of IRS-1- and IRS-2-mediated signaling (37)
.
Thus, in addition to metabolic signals, IRS-3 seems to be able to
mediate mitogenic signals.
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IRS-4 STRUCTURE AND FUNCTION
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IRS-4 is the last member of the IRS family to be identified; it
was initially detected as a 160-kDa protein in human embryonic kidney
(HEK) 293 cell line that was rapidly tyrosine-phosphorylated in
response to insulin but was immunologically distinct from IRS-1
(38)
. Cloning of human IRS-4 has revealed a 1257 amino
acid protein that possesses the typical architecture of the other three
members of the IRS family consisting of a PH domain, a PTB domain, and
a COOH-terminal domain (Fig. 1)
(7)
. The human IRS-4 gene
is localized on the X chromosome. Overall, IRS-4 displays only 27% and
29% sequence identity with IRS-1 and IRS-2, respectively. However, PH
and PTB domains of IRS-4 exhibit a higher degree of homology with the
corresponding IRS-1, IRS-2, and IRS-3 domains. IRS-4 contains 12
potential tyrosine phosphorylation sites, 7 of which lie within the
YXXM motif that binds the SH2 domain of the p85 regulatory subunit of
PI 3-kinase. One potential tyrosine phosphorylation site lies in a
motif that is expected to bind the SH2 domain of Grb-2, and another
site in the motifs known to bind the NH2-terminal
domain of either SHP-2 or phospholipase C. In vitro studies with HEK
cells confirmed that IRS-4 binds PI 3-kinase and Grb-2, but not SHP-2
or phospholipase C (38)
.
Analysis of IRS-4 mRNA distribution by a highly sensitive RT-PCR method
revealed that expression of IRS-4 mRNA is very low but is not limited
to human embryonic kidney 293 cells, where it was originally identified
(8
, 39
, 40)
. Indeed, IRS-4 is expressed in various human
tissues and cell lines including pituitary, thyroid, ovary, prostate,
fibroblasts, IM-9 lymphoblastoid cells, U-2 OS osteogenic sarcoma,
Hep-2 larynx carcinoma cells, A-431 epidermoid carcinoma cells, and
several murine tissues including skeletal muscle, brain, hypothalamus,
liver, heart, kidney, ßTC-1 and ßTC-7 insulinoma cell lines, but
not in the spleen or lung (8
, 39
, 40)
. It has been shown
that differentiation of L6 rat myoblast cells or P19 pluripotent
embryonal carcinoma cells into myotubes was characterized by a marked
increase in IRS-4 mRNA expression, whereas expression of the other IRS
members did not change, thus raising the possibility that IRS-4 may
regulate unique signaling pathways involved in mechanisms of cell
differentiation (8)
. However, IRS-4 is a nonabundant
protein since it cannot be detected in any tissue by standard
immunological methods (39)
. At the subcellular level,
IRS-4 appears to be localized at the plasma membrane (38)
.
The functional role of IRS-4 was investigated by in vitro experiments.
Overexpression of IRS-4 in rat adipocytes led to a marked increase in
the number of GLUT4 molecules recruited to the cell surface
(34)
, whereas overexpression of IRS-4 in 32D hematopoietic
cells lacking any IRS proteins or in NIH-3T3 mouse fibroblasts resulted
in an increased capability of insulin and IGF-I to stimulate cell
proliferation (41
, 42)
. However, in contrast to IRS-1 and
IRS-2, IRS-4 overexpression in 32D cells failed to promote cell
survival, indicating that IRS-4 has different signaling capacity
compared with IRS-1 and IRS-2 (40)
. More recently, it has
been shown that IRS-3 and IRS-4 may act as negative regulators of IGF-I
signaling by suppressing the function of IRS-1 and IRS-2 at several
steps (37)
. Thus, overexpression of both IRS-3 and IRS-4
caused a decrease in IRS-2 mRNA and protein. IRS-3 overexpression also
induced a reduction in IGF-I-stimulated tyrosine phosphorylation of
IRS-1, whereas IRS-4 overexpression decreased the phosphorylation of
both IRS-1 and IRS-2. Finally, overexpression of both IRS-3 and IRS-4
caused a decrease in association of IRS-1 and IRS-2 with the p85
regulatory subunit of PI 3-kinase (37)
. Therefore, the
biological responses to IGF-I in certain tissues may be modulated by a
combination of all four IRS proteins.
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KNOCKOUT MOUSE MODELS LACKING IRSs
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Gene knockout technology has been used widely to investigate the
physiological roles of the IRS family proteins. Knockout mice lacking
IRS-1 (IRS-1-/-) were born alive but exhibited
retardation in embryonal and postnatal growth (16
, 17)
,
indicating that IRS-1 is important for growth-promoting effects of
IGF-I. However, analysis of organ weights indicates organ-specific
differences in the role of IRS-1 as a mediator of growth-promoting
effects of IGF-I (43)
. Thus, skeletal muscle and liver
growth required IRS-1 whereas growth of brain, small intestine, and
spleen did not. Other organs such as kidney and heart showed sexual
dimorphism, since their growth was affected more in females than in
males (43)
. IRS-1-/- mice also
showed resistance to the glucose-lowering effects of insulin and IGF-I,
but had normal fasting glycemia and a mild glucose intolerance from
compensatory hyperinsulinemia caused by selective ß cell hyperplasia.
However, even though ß cell mass was increased in
IRS-1-/- mice, islets from these animals
exhibited defects in insulin content and secretion in response to
glucose (25
, 44)
. These defects are caused by impairment
in the autocrine-activated release of Ca2+ from
intracellular Ca2+ stores, resulting in a
decrease in intracellular free Ca2+ concentration
(45)
. Hyperinsulinemic-euglycemic clamp studies have
clarified the relative contribution of the key insulin-responsive
organs (skeletal muscle, liver, and adipose tissue) to in vivo insulin
resistance. IRS-1-/- mice showed a marked
defect in insulin-stimulated glucose transport and muscle glycogen
synthesis (46
, 47)
. At the molecular level, this defect is
caused by reduced insulin-stimulated PI 3-kinase activity associated
with tyrosine-phosphorylated proteins in skeletal muscle, which are not
compensated by a constitutive increase in the IRS-2 protein content
and, upon insulin stimulation, in its activation (46
, 47)
.
By contrast, insulin-mediated suppression of hepatic glucose production
was relatively unaffected because of full compensation by IRS-2,
resulting in insulin-stimulated PI 3-kinase activity associated with
tyrosine-phosphorylated proteins equivalent to that of wild-type mice
(46
, 47)
. It has been also shown that
IRS-1-/- mice exhibited features of syndrome X,
including hypertriglyceridemia, hypertension, and impaired
endothelium-dependent vascular relaxation (48)
. The
former effect may be caused by both reduced lipoprotein lipase activity
and diminished lipid synthesis in the adipose tissue (21)
.
More recently, it has been reported that
IRS-1-/- mice showed severe osteopenia with low
bone turnover, a characteristic of senile osteoporosis in aged humans
(49)
. Osteoblasts from IRS-1-/-
mice showed defects in proliferation and differentiation induced by
IGF-I and insulin, as well as impairment in the ability to support
osteoclastogenesis. Thus, IRS-1 may play a critical role in maintaining
bone turnover by mediating anabolic actions of insulin and IGF-I.
Unlike IRS-1-/- mice, knockout mice lacking
IRS-2 (IRS-2-/-) exhibit nearly normal birth
size and body weight, but show insulin resistance with abnormal glucose
tolerance at birth and progressively develop fasting hyperglycemia as a
result of inadequate compensatory insulin secretion because of reduced
ß cell mass (44
, 50)
. This event appears to be caused by
defects in ß cell development and increased ß cell apoptosis due to
the failure of IGF-I to promote these effects through IRS-2 signaling
(51)
. Even though ß cell mass was reduced in
IRS-2-/- mice, individual ß cell showed
normal or increased insulin secretion in response to glucose
(44)
. Using a hyperinsulinemic-euglycemic clamp technique,
it has been demonstrated that IRS-2-/- mice
were characterized by a defect in muscle glycogen synthesis, a marked
decrease in the ability of insulin to suppress hepatic glucose
production, and a reduced hepatic glycogen synthesis (46)
.
IRS-2-/- mice also exhibited marked insulin
resistance in adipose tissue as reflected by decreased suppression of
plasma free fatty acid levels and glycerol turnover during the
hyperinsulinemic-euglycemic clamp (46)
. Skeletal muscle
obtained from IRS-2-/- mice showed normal basal
and insulin-stimulated glucose uptake when isolated from animals with
near-normal blood glucose levels (52)
. By contrast,
skeletal muscle isolated from IRS-2-/- mice
that had developed severe hyperglycemia exhibited impaired basal and
insulin-stimulated glucose uptake, suggesting that prolonged
hyperglycemia due to hepatic insulin resistance and ß cell failure,
rather than the lack of IRS-2 in skeletal muscle, was the principal
mechanism for in vivo insulin resistance (44
, 50
, 52)
. At
a molecular level, insulin-stimulated PI 3-kinase activity associated
with tyrosine-phosphorylated proteins was reduced in the liver of
IRS-2-/- mice but normal in their skeletal
muscle (44)
. Accordingly, previous studies with IRS-1
knockout mice or cells derived from these mice have suggested that
IRS-2 could compensate for IRS-1 deficiency more effectively in liver
and pancreatic ß cells than in skeletal muscle, fibroblasts, or
adipocytes (16
, 17
, 20
, 33
, 47)
. Studies in mice with
combined heterozygous knockout of the insulin receptors IRS-1 and IRS-2
suggest that IRS-1 plays a prominent role in skeletal muscle as does
IRS-2 in the liver (53)
.
More recently, it has been shown that IRS-2-/-
females exhibited defects in the hypothalamic-pituitary-ovarian axis
resulting in infertility and moderate obesity (54)
.
Ovaries from IRS-2-/- mice were small and
showed features of anovulatory including decreased number of follicles,
thickening of the cortex and of the stroma, and an almost complete
absence of corpora lutea (54)
. Plasma concentrations of
luteinizing hormone, prolactin and sex steroids were reduced in
IRS-2-/- mice, and these animals were also
resistant to exogenous gonadotropin stimulation. Pituitaries were
reduced in size and contained diminished numbers of gonadotrophs, but
not of somatotrophs. IRS-2-/- females also
showed increased food intake, moderate obesity, and increased body fat
despite elevated leptin levels. These results raise the intriguing
possibility that dysregulation of IRS-2 expression or function may
represent one of the molecular defects responsible for human
infertility associated with insulin-resistant conditions such as
polycystic ovarian syndrome, and will be discussed below.
Taken together, these data suggest that IRS-1 and IRS-2 are not
functionally interchangeable in tissues that are responsible for
glucose production (liver), glucose uptake (skeletal muscle and adipose
tissue), and insulin production (pancreatic ß cells). Thus, IRS-2
seems to have a major role in regulating hepatic insulin action and in
controlling pancreatic ß cell development and survival. By contrast,
in tissues such as skeletal muscle and adipose tissue, defects in IRS-1
signaling cannot be entirely compensated for by IRS-2 (Fig. 2
).
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Figure 2. Schematic diagram depicting the different physiological roles of IRS-1
and IRS-2 as realized by animal and human studies. Note the prevalent
action of IRS-1 in regulating muscle glucose transport, brown adipocyte
differentiation, and insulin-induced ß cell insulin secretion; a role
in bone turnover is demonstrated. IRS-2 appears to play a crucial role
in regulation of pancreatic ß cell development and differentiation,
fat lipolysis, hepatic glucose production, and pituitary–ovarian axis
function.
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Knockout mice lacking IRS-3 exhibited normal growth throughout
development, normal glucose and insulin levels in the fed and fasted
state, normal glucose tolerance, as well as normal basal and
insulin-stimulated glucose transport in isolated adipocytes, thus
arguing against a major role of this substrate in mediating biological
signals of insulin (55)
.
Knockout mice lacking IRS-4 exhibited slightly reduced weight, slightly
lower glucose levels associated with normal plasma insulin levels, and
a slightly impaired oral glucose tolerance test but a normal
intraperitoneal insulin tolerance test (56)
. Female mice
lacking IRS-4 were less fertile than the wild-type. Because IRS-4 is
expressed in organs important for reproduction such as hypothalamus,
pituitary, and ovary, it is tempting to speculate that IRS-4 may be
involved in controlling fertility. Overall, the results of in vivo
studies are not supportive of a major physiological role for IRS-4 in
controlling both growth and glucose metabolism.
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IRS EXPRESSION AND FUNCTION IN HUMANS
|
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Defects in IRS expression and function have been reported in
target tissues of insulin action from insulin-resistant subjects. In
skeletal muscle strips from morbidly obese subjects, it was observed a
significant reduction in IRS-1 content, insulin-stimulated IRS-1
phosphorylation, and PI 3-kinase activation that was paralleled by a
decrease in insulin-stimulated glucose uptake (57)
. A
decrease in insulin-stimulated IRS-1 phosphorylation and PI 3-kinase
activity, but not IRS-1 content, was also observed in skeletal muscle
obtained from nonobese type 2 diabetic patients during a modified
hyperinsulinemic clamp (58)
. Accordingly, in skeletal
muscle biopsies obtained from obese type 2 diabetic patients before and
at the end of a 3 h hyperinsulinemic-euglycemic clamp, a decrease
in both IRS-1- and IRS-2-associated PI 3-kinase activity was observed
(59)
. Impairment in insulin-stimulated phosphorylation of
IRS-1 has been described in skeletal muscle from pregnant obese women
with and without gestational diabetes (60)
. This defect
was primarily due to decreased expression of IRS-1 protein, whereas
IRS-2 expression appeared to be increased and correlated significantly
with impairment in glucose transport (60)
. A similar shift
from IRS-1 to IRS-2 expression has been observed in ovaries from women
with gestational diabetes and polycystic ovary syndrome
(61)
. In women with normal ovulation, IRS-1 was the main
insulin substrate with wide distribution in all compartments of the
follicle including the oocyte, granulosa, thecal cells, and stromal
cells. By contrast, IRS-2 expression is restricted to the theca
interna. Furthermore, expression of IRS-1 increased during follicular
development whereas IRS-2 remained unchanged. In women with gestational
diabetes or polycystic ovary syndrome, expression of IRS-2 increased in
oocytes, granulosa cells, and theca internal cells whereas IRS-1
expression decreased in the granulosa cells (61)
. Because
IRS-2 was shown to be an antiapoptotic mediator (62)
,
overexpression of IRS-2 in antral follicles may affect their
physiological apoptosis resulting in later accumulation of larger
cysts, a typical feature of polycystic ovary syndrome. A reduction in
IRS-1 expression has been also reported in isolated adipocytes from
type 2 diabetic patients (18)
. In these cells, IRS-2
levels remained unchanged but did not fully compensate for IRS-1
down-regulation (18)
. Low IRS-1 gene and protein
expression in adipocytes has been described in 30% of subjects at high
risk for type 2 diabetes, i.e., first-degree relatives of type 2
diabetics and morbidly obese subjects (63)
. Moreover,
subjects with low IRS-1 expression were characterized by clinical
features of the insulin resistance syndrome including higher fasting
glucose and insulin levels, higher triglyceride levels, and higher
visceral adiposity measured by waist/hip ratio (63)
.
Overall, these results indicate that low expression of IRS-1 in target
tissues of insulin action (i.e., skeletal muscle and adipose tissue)
may be considered a molecular marker of insulin-resistant states such
as obesity and type 2 diabetes.
|
IRS POLYMORPHISMS IN TYPE 2 DIABETES
|
|---|
IRS-1 polymorphisms in type 2 diabetes
Molecular scanning of the IRS-1 gene in more than 4,000 normal
individuals and patients with type 2 diabetes from different ethnic
groups has revealed several polymorphisms resulting in amino acid
substitutions (Table 2
) (64
65
66
67
68
69
70
71
72
73
74
75
76
77
78)
. Overall, the frequency of IRS-1 polymorphisms
is higher in type 2 diabetic patients than in control subjects. Of
these, the GlyArg change at codon 972 (Arg972)
IRS-1) is the most common, and has been studied most extensively
(64
65
66
67
68
69
70
71
72
73
74
75
76
77
78)
. Several reports have indicated a higher
prevalence of the Arg972 polymorphism in type 2
diabetic patients (64
65
66
67
68)
, although other studies have
reported a weak (69
70
71
72
73)
or absent (74
–58
)
association between this variant and type 2 diabetes. Possible
explanations for these divergent results may lie in the ethnic
differences in the genetic background for type 2 diabetes. For example,
the prevalence of the Arg972
polymorphism in both diabetic and control Japanese populations appears
to be lower than that observed in the corresponding Caucasian
populations (3.8 vs. 10.6% in type 2 diabetes, and 4.0% vs. 6.5% in
control subjects, respectively). In Japanese populations, however,
insulin sensitivity (measured by hyperinsulinemic-euglycemic clamp) is
decreased by 22% in normal and by 29.5% in type 2 diabetic subjects
with the Arg972 polymorphism vs. those in
comparable groups without polymorphism (71)
. Curiously,
the Arg972 polymorphism is absent in Pima Indians
(78)
.
Besides ethnic differences, significant regional differences in the
prevalence of the Arg972 polymorphism have been
observed between Dutch nondiabetic subjects from Hoorn and Rotterdam
(9.4 vs. 18.6%; P<0.05) (76)
, thus
complicating the interpretation of association study of this IRS-1
variant. Another possible explanation may lie in the number of subjects
examined in a single study. Using exact permutational methods, the
number of subjects needed to achieve a conventional level of
significance, assuming the Arg972 polymorphism is
present in 10% of type 2 diabetic patients and 5% of control
subjects, has been estimated (66)
. To detect an
association with a P value of 0.01, 380 diabetic and 380
control subjects would need to be analyzed. Therefore, most published
studies by themselves lack the power to determine an association
between the Arg972 variant and type 2 diabetes.
By contrast, when all available studies in Caucasian populations are
pooled, an overall association between type 2 diabetes and the
Arg972 polymorphism ensues (66
, 68)
.
Finally, because type 2 diabetes is a heterogeneous disorder, the
Arg972 IRS-1 polymorphism may act in combination
with environmental factor such as diet, physical activity, and age to
confer susceptibility to diabetes. Consistent with this idea, the
Arg972 polymorphism of IRS-1 in its
heterozygous form is associated in obese nondiabetic subjects with a
50% reduction in insulin sensitivity vs. obese subjects without
polymorphism, indicating that the polymorphism potentiates
obesity-linked insulin resistance (79)
. These obese
carriers of the Arg972 IRS-1 polymorphism are
also characterized by a clustering of metabolic cardiovascular risk
factors with elevated fasting levels of plasma glucose, serum
triglyceride, plasma tissue-plasminogen-activator, and its inhibitor
PAI-1, which suggests that the polymorphism potentiate obesity-linked
insulin resistance (79)
. In a group of subjects selected
from the UK Prospective Diabetes Study (UKPDS), the prevalence
Arg972 IRS-1 polymorphism was increased in
subjects with type 2 diabetes who had insulin resistance associated or
not with dyslipidemia (67)
. More recently, a 2.8-fold
higher frequency of the Arg972 IRS-1 polymorphism
has been reported in patients with angiographic evidence of coronary
artery disease (CAD) than in control individuals (80)
.
When adjusted for other risk factors, the relative risk of CAD
associated with the Arg972 IRS-1 polymorphism was
2.93-fold higher than in wild-type individuals, and it increased to
6.97-fold in obese subjects and to 27.3-fold in subjects with clinical
features of insulin resistance syndrome (80)
. The
Arg972 IRS-1 polymorphism was also associated
with a higher frequency of diabetes, hypertriglyceridemia, and
hypercholesterolemia (80)
. However, these results have not
been confirmed in another study of 169 overweight/obese patients with
type 2 diabetes (73)
. Possible explanations for these
apparent contradictions may lie in ethnic differences, different degree
of insulin resistance, interaction with other metabolic factor, size of
examined population sample. The Arg972 IRS-1
variant was not associated with decreased birth weight, which has been
proposed as a risk factor for insulin resistance and type 2 diabetes
(81)
. Diabetic patients with the
Arg972 IRS-1 variant are further characterized by
lower plasma levels of fasting insulin and C-peptide (64)
.
In addition, glucose-tolerant subjects who were heterozygous for the
Arg972 variant exhibited a lower insulin response
to an oral glucose load and decreased insulin secretion during a
hyperglycemic clamp compared with noncarriers (82)
. Taken
together, these observations raise the possibility that the
Arg972 IRS-1 polymorphism in combination with
both defects in other candidate genes and environmental factors may
account for the phenotype of peripheral insulin resistance and impaired
insulin secretion, the two typical features of type 2 diabetes.
A series of in vitro studies has been carried out to investigate the
functional effects of the GlyArg change at codon 972 on insulin
action and insulin secretion (15
, 24
, 83
84
85)
. Expression
of the Arg972 IRS-1 variant in 32D-IR cells
caused a specific defect in binding of the p85 regulatory subunit of PI
3-kinase to the IRS-1 variant and a 36–39% decrease in
IRS-1-associated PI 3-kinase activity (83
, 84)
. The
GlyArg972 change did not alter the level of
expression of IRS or the extent of insulin-stimulated tyrosine
phosphorylation of IRS-1, consistent with the idea that the
Arg972 IRS-1 variant interfered with the
interaction between IRS-1 and the SH2 domains of PI 3-kinase, possibly
by altering the tertiary structure of IRS-1. The net effect was a 32%
decrease in the mitogenic effects of insulin (83
, 84)
.
More recent studies have addressed the question of the functional
significance of the Arg972 IRS-1 variant on
insulin-dependent glucose metabolism (15)
. Expression of
the Arg972 IRS-1 variant in L6 skeletal muscle
cells resulted in a decrease in both basal and insulin-stimulated
glucose transport compared with L6 cells expressing wild-type IRS-1
(15)
. These alterations were associated with a reduction
in the amount of GLUT4 translocated to the plasma membrane under both
the basal condition and in response to insulin, without changes in
total cellular GLUT4 protein content. The molecular mechanism by which
the Arg972 IRS-1 variant affected glucose
metabolism was further elucidated by experiments examining downstream
targets in PI 3-kinase signaling pathway. There is evidence that the
Ser/Thr kinase Akt, a downstream effector of PI 3-kinase, acts as a key
enzyme linking PI 3-kinase activation to multiple biological function
of insulin, including glucose transport and glucose transporters
translocation to the plasma membrane (86)
. Consistent with
this idea, expression of the Arg972 IRS-1 variant
in L6 myocytes resulted in a significant decrease in Akt
phosphorylation and activity because of defective IRS-1-associated PI
3-kinase (15)
. Akt has been also implicated in the
regulation of glycogen synthesis through a mechanism involving
phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3)
that results in increased activity of the active dephosphorylated form
of glycogen synthase. Expression of the Arg972
IRS-1 variant in L6 myocytes resulted in a significant decrease in
GSK-3 phosphorylation and inactivation by Akt; these defects are
associated with a reduction in insulin-stimulated glucose incorporation
into glycogen and glycogen synthase activity (15)
.
Overall, these results suggest that the Arg972
IRS-1 polymorphism might contribute to the development of insulin
resistance by impairing the ability of insulin to activate the IRS-1/PI
3-kinase/Akt/GSK-3 signaling pathway, thus leading to defects in
glucose transport, glucose transporters translocation and glycogen
synthesis.
As reported above, diabetic and prediabetic carriers of the
Arg972 IRS-1 variant are characterized by a low
fasting plasma concentration of insulin and C-peptide (64
, 82)
. There is increasing evidence that insulin synthesis and
secretion may be modulated by autocrine activation of the insulin
signaling involving insulin receptor phosphorylation, tyrosine
phosphorylation of IRS-1, and activation of PI 3-kinase
(23
24
25)
. These observations have led us to hypothesize
that the Arg972 IRS-1 polymorphism may affect
pancreatic ß cell insulin secretion. Expression of the
Arg972 variant in RIN rat ß cell line resulted
in a 60% decrease in binding of the p85 subunit of PI 3-kinase to
IRS-1 and a 50% decrease in IRS-1-associated PI 3-kinase activity,
whereas expression or function of endogenous IRS-2 was not affected
(24)
. These changes caused a marked decrease in insulin
secretory response to glucose and glibenclamide (24)
(Fig. 3
). A more recent study has addressed the role of
Arg972 IRS-1 variant in human pancreatic islet
function and survival (87)
. The results obtained document
that pancreatic islets isolated from carriers of
Arg972 IRS-1 exhibited impaired IRS-1-associated
PI 3-kinase activity, reduced insulin secretion, and increased
apoptosis and were resistant to the antiapoptotic effect of insulin
compared with wild-type controls. The same results were reproduced in
the RIN rat ß cell line stably expressing either wild-type IRS-1 or
Arg972 IRS-1 variant. A defective activation of
the PI 3-kinase/Akt survival pathway resulting in lower phosphorylation
and inactivation of the proapoptotic protein Bad, a member of the Bcl-2
family of proteins known to play a central role in the regulation of
cell death, appeared to be the mechanism responsible for the increased
apoptosis observed in pancreatic islets isolated from carriers of
Arg972 IRS-1 (87)
.
View larger version (56K):
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|
Figure 3. Schematic diagram of the functional effects of the Arg972
IRS-1 variant on insulin secretion and action.
|
|
Several additional polymorphisms resulting in amino acid substitutions
have been described, but their frequency is lower than that of the
Arg972 IRS-1 variant (Table 2)
. The
SerGly893 IRS-1 variant has been detected in 3
of 112 Finnish patients with type 2 diabetes and in 1 of 104 control
subjects (72)
. Although the functional impact of the
SerGly893 change has not been studied in
vitro, this site is located immediately upstream to the SH2 binding
site of Grb-2, an adaptor protein that links IRS-1 to the mitogenic
Ras/MAP kinase pathway. Two other IRS-1 polymorphisms—the
GlyArg819 and the
ArgCys1221—were found in the same subject who
was also heterozygous for the Arg972 IRS-1
polymorphism (65
, 85)
. Although the
Cys1221 is located between two potential sites of
tyrosine phosphorylation (Tyr1179 and
Tyr1229) that are thought to be involved in
binding of the tyrosine protein phosphatase SHP-2, neither of the two
IRS-1 polymorphisms affected expression or function of IRS-1 when
expressed in Cos7 cells (85)
. In Japanese type 2 diabetic
patients, three additional polymorphisms have been described, including
the ProArg170,
MetThr209, and
SerPhe809 (71)
. Two of these,
Arg170 and Thr209, are
located in the PTB domain of IRS-1, and the nonconservative
substitutions of the native residues were expected to affect the
interaction of IRS-1 with the insulin receptor. Consistent with this
hypothesis, expression of the Arg170 and the
Thr209 IRS-1 variant in 32D-IR cells both
resulted in a reduced binding to the insulin receptor on insulin
stimulation leading to a decrease in tyrosine phosphorylation of IRS-1,
PI 3-kinase activation, MAP kinase activity, and DNA synthesis
(84)
. Although the functional significance of the
SerPhe809 IRS-1 variant has not been studied
in vitro, this polymorphism has been detected only in type 2 diabetic
subjects (71)
. In addition, diabetic carriers of the
Phe809 IRS-1 variant exhibited higher HBA1c,
fasting glucose, and postprandial glucose levels than diabetic
noncarrier (50)
. Phenotype analysis of type 2 diabetic
patients who are heterozygous for the Pro513
IRS-1 polymorphism did not reveal any significant differences compared
with wild-type diabetic subjects, which argues against a major
pathogenetic role of this IRS-1 variant (64
, 67)
.
IRS-2 polymorphisms in type 2 diabetes
In contrast to the phenotype of IRS-2-/-
mice, polymorphisms of the human IRS-2 gene did not appear to
contribute to the pathogenesis of either the common form of type 2
diabetes or early-onset autosomal dominant type 2 diabetes (Table 1)
(26
, 88
89
90
91)
. No genetic variability has been identified
in the promoter sequence of IRS-2 in type 2 diabetic patients, which
suggests that mutations causing alterations in IRS-2 expression levels
are rare (89)
. A relatively common IRS-2 polymorphism
causing the GlyAsp change at codon 1057 has been detected in a
Danish population. However, its allelic frequency was similar in both
normal subjects (33.9%) and type 2 diabetic patients (33.8%)
(88)
. A much less frequent IRS-2 polymorphism that causes
the GlySer change at codon 879 has been also detected in the same
population and shows no differences in frequency between normal and
diabetic subjects (88)
.
The potential effect of the Asp1057 IRS-2 variant
on insulin secretion and insulin sensitivity has been examined in four
groups of glucose-tolerant Scandinavian subjects (89)
.
This polymorphism had no detectable effect on insulin secretion and
insulin sensitivity in a group of 363 young healthy Danish subjects, in
a group of 228 Danish glucose-tolerant offspring of one type 2 diabetic
parent or in a cohort of 639 glucose-tolerant elderly Swedish men. In
contrast, examination of 236 middle-aged glucose-tolerant Danish
subjects showed that homozygous carriers of the
Asp1057 IRS-2 variant were characterized by a
25% decrease in fasting insulin levels and a 17% decrease in fasting
C-peptide levels compared with wild-type carriers (89)
.
The serum insulin and C-peptide concentrations of the homozygous
carriers of the Asp1057 IRS-2 variant remained
decreased during an oral glucose tolerance test. Despite the reductions
in serum insulin and C-peptide concentrations, plasma glucose levels
were not altered in homozygous carriers of the
Asp1057 IRS-2 variant vs. wild-type carriers
(89)
.
The lack of an overall association between the
Asp1057 IRS-2 variant and type 2 diabetes has
been also reported in an Italian population (91)
.
Curiously, in subjects with BMI < 27 kg/m2,
a lower prevalence of the homozygous Asp1057
IRS-2 genotype was observed in patients with type 2 diabetes compared
with control individuals. By contrast, in subjects with BMI > 27
kg/m2, a higher prevalence of the homozygous
Asp1057 IRS-2 genotype was observed in patients
with type 2 diabetes vs. control individuals (91)
. Thus,
overweight seems to modify the effect of this polymorphism toward a
higher risk of type 2 diabetes. A rare IRS-2 polymorphism that causes
the LeuVal change at codon 647 has been identified in 3 of 413
Danish patients with type 2 diabetes and in none of 280
glucose-tolerant subjects (89)
. Analysis of the family of
one of the carriers of the Val647 IRS-2 variant
showed diabetes in the parents and one brother, and impaired glucose
tolerance in another brother (89)
. The
Val647 IRS-2 variant is located in the KRLB
domain, which in addition to the PH and PTB domains, is involved in the
interaction with the insulin receptor ß subunit (11)
.
The Val647 IRS-2 variant is close to the
Tyr653 residue in a YMXM motif, which is a
binding site for the p85 regulatory subunit of PI 3-kinase. However,
expression of the Val647 IRS-2 variant in the
yeast two-hybrid system did not affect interaction of the IRS-2 KRLB
domain with either the insulin receptor or p85 subunit of PI 3-kinase.
Overall, the data indicate that polymorphisms in IRS-2 gene do not have
consistent functional effects on insulin secretion or insulin
sensitivity in type 2 diabetes.
Other IRS polymorphisms in type 2 diabetes
Because the sequence of human IRS-3 gene is unknown, there is no
information on whether polymorphisms in IRS-3 may contribute to the
pathogenesis of type 2 diabetes. By contrast, five polymorphisms in the
IRS-4 gene have been identified in Danish Caucasian subjects (Table 1)
(92)
. The allelic frequency of these IRS-4 polymorphisms
has been determined in 324 type 2 diabetic patients and in 267 control
subjects. Two amino acid variants (the
GlyCys584 and the
LysThr883) are rare; each has been found in
the heterozygous form in only one type 2 diabetic patient. The allelic
frequency of the other three polymorphisms
(LeuPhe34, ArgGly411,
and HisAsp879) is similar in both normal
subjects and type 2 diabetic patients. The most prevalent IRS-4 variant
is the HisAsp879, which displays an allelic
frequency of 19.2% in type 2 diabetic patients vs. 18.0% in control
subjects (92)
. Clinical and biochemical characteristics of
heterozygous and homozygous subjects carrying either the
ArgGly411 or
HisAsp879 IRS-4 variant are indistinguishable
from those of wild-type IRS-4 carriers (92)
. Thus, these
data indicate that polymorphisms in IRS-4 are common in Caucasian
populations but are not associated with type 2 diabetes or insulin
resistance, consistent with the failure of IRS-4 knockout mice to
result in a diabetic phenotype (92)
.
|
CONCLUSIONS
|
|---|
The role of IRS proteins as key mediators of insulin signaling is
well established. It is less clear whether the four members of the IRS
family play redundant or specific roles in insulin signal transduction.
Although the IRS molecules are phylogenetically conserved and share
structural similarities, there are differences in phosphorylation
motifs, tissue distribution, subcellular localization, developmental
expression, binding to the insulin receptor, and interaction with SH2
domain-containing proteins. These differences may provide a potential
mechanism for signaling diversity by the four molecules through the
formation of specific signaling complexes in different intracellular
sites of different tissues.
The search for specific genetic defects that contribute to the main
defects in type 2 diabetes, i.e., insulin resistance and insulin
deficiency, is complicated by the fact that this syndrome is polygenic
with complex inheritance patterns. Although considerable effort has
been devoted to identifying potential disease-causing mutations in
candidate genes, no single major susceptibility gene for the common
form of type 2 diabetes has been identified. The most likely
explanation for these disappointing results is that type 2 diabetes
consists of several subtypes of the disease, where a combination of
different mechanisms (including genetic and environmental factors) acts
synergistically to impair insulin secretion and action. Thus, the
modest association between the Arg972
polymorphism of IRS-1 and type 2 diabetes suggests that this relatively
common mutation may contribute to the pathogenesis of type 2 diabetes
in a small subgroup of cases. Furthermore, it is possible that the
Arg972 IRS-1 variant is not a sufficient cause of
diabetes, but interacts with environmental factors to impair insulin
sensitivity. In addition, a combination of two or more mild genetic
defects in insulin signaling may account for the multifactorial nature
of the disease, as demonstrated in transgenic mice with simultaneous
disruption of two distinct genes such as IRS-1 and insulin receptor
(93)
or IRS-1 and glucokinase (94)
. As the
molecular mechanisms regulating insulin secretion and insulin action
unfold, we will have increasing possibilities to elucidate the role of
specific insulin signaling elements in insulin action and ß cell
function and to design efficacious, safe, and convenient interventions.
The study of animals with targeted disruption of genes encoding the
four IRS proteins has provided important clues to the
pathophysiological role of these signaling molecules.
|
ACKNOWLEDGMENTS
|
|---|
This work was supported in part by grants from European Community
no. QLG1-CT-1999–00674 (G.S.) and Telethon-Italy no. E.695 and E.1309
(G.S.), Progetto di Ricerca Finalizzata RF98 from Ministero della
Sanità (G.S. and M.F.), PRIN-COFIN 1999 no. 9906277284–001 and
9906277284–002 from Ministero dell’Università e Ricerca
Scientifica e Tecnologica (R.L. and G.S.).
|
REFERENCES
|
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De Fronzo, R. A. (1997) Pathogenesis of type 2 diabetes: metabolic and molecular implications for identifying diabetes genes. Diabetes Rev 5,177-269
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White, M. F. (1997) The insulin signaling system and the IRS proteins. Diabetologia 40,S2-S17
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Virkamaki, A., Ueki, K., Kahn, C. R. (1999) Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J. Clin. Invest. 103,931-943[Medline]
TOP
ABSTRACT
INTRODUCTION
